Zika virus vaccine and methods of production

ABSTRACT

The invention generally relates to a purified inactivated Zika virus (ZIKV), methods for producing the purified inactivated ZIKV, immunogenic compositions and vaccines comprising the purified inactivated ZIKV and methods for the prevention and/or treatment of infection by ZIKV.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/343,315 filed May 31, 2016 and U.S. Provisional Application No.62/370,260 filed Aug. 3, 2017, both of which are incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was created with U.S. Government funding. The U.S.Government has rights in this invention.

FIELD

Described are immunogenic compositions, vaccines, and methods forimmunization and protection (e.g., prophylaxis) against Zika virus(ZIKV) infection, treatment of ZIKV infection and symptoms, andimmunization/protection/treatment of associated diseases, and clinicalconditions. Also provided are purified, inactivated ZIKV compositionscomprising virus that is re-derived from a ZIKV strain, and whichconfers an antibody titer sufficient for broad-based seroprotectionagainst all strains of ZIKV.

BACKGROUND

Zika virus (ZIKV) is a member of the Flaviviridae virus family and theflavivirus genus. In humans, it causes a disease known as “Zika”. It isrelated to dengue, yellow fever, West Nile and Japanese encephalitisviruses that are also members of the virus family Flaviviridae. Alongwith other viruses in this family, ZIKV is enveloped and icosahedralwith a non-segmented, single-stranded, positive sense RNA genome. ZIKVis transmitted by mosquitoes and has been isolated from a number ofspecies of the Aedes genus. The virus was first isolated in 1947 from arhesus monkey in the Zika Forest of Uganda, Africa, and was isolated forthe first time from humans in 1952 in Nigeria. Evidence of humaninfection has been reported from other African countries as well as inparts of Asia including India, Malaysia, the Philippines, Thailand,Vietnam, and Indonesia. In 2007 there was a Zika outbreak on theMicronesian Island of Yap, in 2013 there was an estimated 28,000 Zikacases in French Polynesia, and since early 2015 ZIKV has been infectingtens and perhaps hundreds of thousands in the Americas driving hundredsof travel related cases globally. Common symptoms of infection with ZIKVinclude mild headaches, maculopapular rash, fever, malaise,conjunctivitis, and arthralgia. Available data indicate approximately20% of those infected will develop this mild disease phenotype. Therecent outbreaks in French Polynesia and the Americas reveal thepotential for very serious and sometimes fatal outcomes followingcongenital ZIKV infection (microcephaly) and serious neurologic sequelaefollowing infection (Guillaing Barrè syndrome (GBS)) (22). Researchersestimate between 1-13% of ZIKV infections during the first trimester ofpregnancy will result in microcephaly. In addition, ZIKV has beencausally associated with intrauterine growth retardation, and othercongenital malformations in both humans (15-18) and mice (19-21).Several reports have shown that ZIKV can infect placental and fetaltissues, leading to prolonged viremia in pregnant women (23) andnonhuman primates (24). ZIKV also appears to target cortical neuralprogenitor cells (19-21, 25 and 26), which likely contributes toneuropathology. The increased incidence of microcephaly temporallyrelated to the introduction of ZIKV in the Americas resulted in theWorld Health Organization establishing a Public Health Emergency ofInternational Concern (PHEIC). It is also because of these severeoutcomes that a protective vaccine is required.

Currently there are no approved or licensed vaccines—or any knowneffective vaccines—to prevent ZIKV infection or disease. A number ofZika vaccine candidates in discovery and pre-clinical stages have beenreported as “under development” in the public domain at scientificmeetings. A search in PubMed using terms “Zika” and “vaccine” (accessed29 May 2016) yielded 80 citations none of which described animal orhuman Zika vaccine data. The World Health Organization conducted areview of the Zika vaccine field (3 Mar. 2016) and concluded there wereup to 18 active programs pursuing a number of different approaches toinclude purified inactivated (none others successful at that point),nucleic acid based vaccines (DNA, RNA), live vectored vaccines, subunitvaccines, VLP technologies and live recombinant approaches. There is anurgent need for a vaccine capable of inducing a protective immuneresponse against infection from ZIKV, as well as compositions to treatZIKV post-infection. Mechanisms of action of a prophylactic vaccinecould include protecting against infection (failure of mosquitotransmitted virus to replicate in the human abrogating infection),protecting against disease (mosquito transmitted virus replicates but atan insufficient level to cause disease), protecting against adverseneurologic sequelae (infection occurs but vaccine induced immunityimpacts rate of adverse outcomes following infection), and/or protectingthe fetus during maternal infection (infection during pregnancy occursbut vaccine induced immunity prevents fetal infection from taking hold).If vaccine-induced immunity kinetics are robust and rapid it isconceivable active immunization AFTER infection may be able to abrogatethe adverse outcomes of ZIKV exposure (congenital, neurologic) beforethey take hold.

SUMMARY OF THE DISCLOSURE

A novel purified, inactivated ZIKV is described, including compositionsand vaccines comprising it, methods for producing the same, and methodsof using the same (e.g., generating an immune response in a subject atrisk of infection and/or in need of preventative treatment, or raisingantibodies in a subject, or alleviating symptoms of ZIKV in an infectedsubject, and the like).

ZIKV can be purified to be free of pathogens and adventitious agents.The ZIKV infectious virus particle can be purified away from the hostcell proteins (demonstrated by known procedures such as gradientcentrifugation and column chromatography). The level of purity isaccording to the guidelines of the U.S. Food and Drug Administration(FDA). The purified ZIKV is then inactivated using known chemical agentsor any treatment which sufficiently preserves viral antigenicity andimmunogenicity while destroying viral infectivity. The resultantpurified, inactivated ZIKV is suitable for use as a vaccine component togenerate an immune response but non-infective for ZIKV, or is part of acomposition that can be used to generate antibodies in a subject exposedto it. It is quite safe for human and mammalian use. Other uses of thepurified, inactivated ZIKV are described below.

The purified, inactivated ZIKV, when used in immunogenic compositionsand vaccines, has a major advantage over attenuated virus vaccines andimmunogenic compositions in that inactivated viruses are not infectiousand therefore, cannot revert to virulence or cause disease. This isimportant when considering vaccination in known or potentially specialpopulations such as those with immunodeficiencies (e.g., HIV) or thosewho are pregnant. Another advantage of inactivated over attenuatedviruses is their potentially greater physical stability (e.g., totemperature changes) allowing easy and economical transport and storageof the vaccine and immunogenic compositions. In addition, inactivatedviruses afford superior immunogenicity and greater protection againstdisease due to their preserved native conformation.

One composition can be a purified-inactivated vaccine (PIV) for ZIKV.The vaccines comprise, consist essentially of, or consist of one or moreof the purified-inactivated ZIKV as described herein. The purified,inactivated ZIKV can be used to immunize mammals (including humans) toelicit high titers of virus neutralizing antibodies and protect theimmunized mammal from disease caused by ZIKV. For example, a singleimmunization of the vaccine can be shown to provide 100% completeprotection in susceptible mammals against challenge with ZIKV. Anotherexample is wherein two doses are administered (e.g., a first dose,followed by a second dose 4 weeks later). A booster dose (second dose ormore) may be useful for persons with recurrent infection risk. This issimilar to the booster dose regimen associated with the PIV for theflavivirus, Japanese encephalitis (JEV). With the information knownabout the dosing and schedule for other PIV flavivirus vaccines—such asJapanese encephalitis and tick-borne encephalitis—someone skilled inthis art can determine a safe and effective dose and schedule for thisZIKV PIV, as needed for persons of any age and size. Furthermore,potential immunologic correlates of protection are shown by the databelow. The vaccine can be suitable for rapid immunization with thepotential to break the cycle of viral transmission at the individual andpopulation levels.

Optionally, this ZIKV vaccine (and any other compositions describedherein) can be mixed with suitable adjuvants.

Compositions can comprise, consist essentially of, or consist of one ormore of the novel purified, inactivated ZIKV as described herein. Theseare immunogenic compositions that are able to produce an immune responsein a mammal—that is, they are able to induce the production ofantibodies which recognize ZIKV, or are reactive with ZIKV.

The term “immunogenic” as used herein has its accepted well-knownmeaning in this art, relating to or denoting substances able to producean immune response, the property enabling a substance to provoke animmune response, or the degree to which a substance possesses thisproperty of immunogenicity. To that end, a composition can contain oneor more of the purified-inactivated ZIKV as described herein, and anadjuvant, such as a pharmaceutical adjuvant. These compositions can beuseful in methods to produce antibodies which recognize ZIKV in a host,when the compositions are administered by known means to the host.

A purified-inactivated ZIKV can be an inactivated strain of the purifiedPuerto Rican strain PRVABC59, described below. The purified-inactivatedZIKV can be any inactivated and purified strain of Zika. The methods forproduction and use as a vaccine would be applicable to strains otherthan the Puerto Rican strain. Additional strains are known currently,and some are described below.

Also provided is a method for producing purified, inactivated ZIKV foruse in any of the vaccines and immunogenic compositions describedherein. The method minimally includes the steps of purifying a selectedZIKV strain, and inactivating it. The method can include the followingsteps are:

(i) inoculating a cell culture with a ZIKV strain;(ii) propagating the virus in the inoculated cell culture;(iii) harvesting and isolating virus fluids from the inoculated cellculture to prepare a ZIKV concentrate; and preferably reducing thepresence of host cell DNA, for instance by treatment of the harvest witha chemical agent such as benzonase;(iv) purifying the ZIKV concentrate;(v) inactivating the purified ZIKV; and(vi) recovering the inactivated purified ZIKV.Also provided is a purified, inactivated ZIKV generated by this method.

A ZIKV strain can be one that has first been subjected to passagingthrough an appropriate cell line—e.g., inoculating a cell culture withthe strain, propagating the virus, harvesting the virus, and clarifyingit. An appropriate cell line is any one that will permit adequate growthof the virus, and produce a viral product suitable for human use. Forexample, Vero cells are very suitable. Passaging cells at least 3 timesproduces an effective starting ZIKV strain (or “Master Seed”), althoughfewer or more passages can be done. The Master Seed can be frozen andtested and used for the purification-inactivation process describedherein. In one embodiment described herein, where a Master Seed isproduced by three passages, the purification steps (i)-(iii) of ourprocess can be referred to as the fourth passage. The purificationprocess can further include a re-derivation of the ZIKV strain by RNAtransfection. In the preparation of the Master Seed, at the end of asecond passage, RNA can be extracted and used in a third passage fortransfection. This can be done using standard methods, as the strain ispropagated in a cell line. This method results in reproducing a cleancopy of the Zika virus within the cell line, and in so doing, removespossible contaminating adventitious agents, creating a purer strain.This RNA rederivation process reduces the risk of “carry-over”adventitious agents.

Inactivation can be done by contacting the purified ZIKV with a chemicalinactivating agent, such as formalin or beta-propiolactone, orcombinations of these, and other known agents.

The purified ZIKV strain PRVABC59 passaged 3 times is a particularlyuseful master seed that could be used for multiple vaccine lotproductions at passage 4 in the purification-inactivation method.PRVABC59p-3 is a unique Master Seed, having a unique sequence andproperties.

The methods described herein also entail using host cells that areuseful for ZIKV vaccine production strains. An exemplary host cell lineis Vero cells, although any cell line could be used that is permissivefor growth of the ZIKV and yields product that is useful for ultimatehuman use.

Also contemplated are methods and kits to induce immune responses toZIKV, or raise antibodies that recognize ZIKV, in a mammal (especiallyhumans). The method comprises administering to a subject a compositioncomprising, including or consisting essentially of, one or more of thepurified-inactivated ZIKV as described herein, in a pharmaceuticallyacceptable adjuvant, in an amount effective to cause an immune response(including raising antibodies that recognize ZIKV) in the subject. Thecomposition may be a vaccine, and the immune response may be aprotective immune response. Specifically, methods are provided forimmunizing a mammal (especially a human) against ZIKV infection, whichcomprises administering to the mammal an amount of one or more of thevaccines disclosed herein to achieve effective immunization againstZIKV. Booster doses may be used, if needed. Administration may be by anyknown route, such as transcutaneous injection, intramuscular injection,intradermal injection, subcutaneous injection, intravenous injection,oral, or intranasal inoculation. A kit would contain one or more of thecompositions described herein, and can include instructions for use.

Other uses of the purified, inactivated ZIKV as described herein includealleviating symptoms of ZIKV and/or treatment of ZIKV infection (e.g.,post-infection). For example, in some vaccine embodiments, the ZIKVvaccine is effective to protect against disease prior to ZIKV exposureand infection, as well as alleviate disease and clinical symptomsassociated with ZIKV following ZIKV exposure.

The method comprises administering to a subject infected with ZIKV acomposition comprising, including or consisting essentially of, one ormore of the purified-inactivated ZIKV as described herein, in apharmaceutically acceptable adjuvant, in an amount effective to cause animmune response (including raising antibodies that recognize ZIKV) inthe subject. The immune response effectively alleviates ZIKV symptoms orotherwise effectively treats ZIKV infection. The composition can beadministered to an infected subject as soon as possible followinginitial infection, or at least between initial infection and developmentof congenital infection or onset of severe symptoms. Ideally, only onedose is needed to effect protection against Zika infection for mammals,including humans. An exemplary dosage schedule entails an initial dose,then a second (booster) dose about 4 weeks later. However, this scheduleis exemplary only, and someone skilled in this art would be able todetermine without undue experimentation if and when any second dose isneeded.

Additional materials and methods are as follows:

-   -   1. A purified inactivated Zika virus (ZIKV).    -   2. A purified, inactivated, immunogenic ZIKV.    -   3. The purified, inactivated, immunogenic ZIKV of claim 2,        wherein the ZIKV is PRVABC59, and the virus is purified and        inactivated.    -   4. An immunogenic composition comprising a purified inactivated        ZIKV and a pharmaceutically acceptable adjuvant.    -   5. An immunogenic composition comprising the purified,        inactivated, immunogenic ZIKV of any of claim 2 or 3 and an        acceptable adjuvant.    -   6. The immunogenic composition of claim 4, wherein the        acceptable adjuvant is alum.    -   7. The immunogenic composition of claim 1, wherein the purified        inactivated ZIKV is derived from ZIKV PRVABC59.    -   8. The immunogenic composition of claim 4, wherein the purified        inactivated ZIKV is derived from ZIKV PRVABC59.    -   9. The immunogenic composition of any of claims 4 to 8, wherein        the purified inactivated ZIKV is derived from Puerto Rico        PRVABC59, Thailand SV0127/14, Philippine COC C 0740, or Brazil        Fortaleza/2015, or other suitable strains.    -   10. A vaccine comprising a purified inactivated ZIKV and a        pharmaceutically acceptable adjuvant.    -   11. A vaccine comprising the purified, inactivated, immunogenic        ZIKV of claim 1A and a pharmaceutically acceptable adjuvant.    -   12. The vaccine of any of claim 10 or 11, wherein the        pharmaceutically acceptable adjuvant is alum.    -   13. The vaccine of claim 10, wherein the purified inactivated        ZIKV is derived from ZIKV PRVABC59.    -   14. The vaccine of claim 11, wherein the purified inactivated        immunogenic ZIKV is derived from Puerto Rico PRVABC59, Thailand        SV0127/14, Philippine COC C 0740, or Brazil Fortaleza/2015, or        other suitable strains.    -   15. A method of producing antibodies which recognize ZIKV in a        host comprising administering to the host a composition        comprising the immunogenic composition of any of claims 4 to 9.    -   16. A method of inducing a protective immune response against a        Zika virus (ZIKV) in a subject, comprising the step of        administering to the subject the vaccine of claim 10.    -   17. A method of inducing a protective immune response against a        Zika virus (ZIKV) in a subject, comprising the step of        administering to the subject the vaccine of claim 11 or 14.    -   18. The method of any of claim 16 or 17, wherein the        administering is via intramuscular injection, intradermal        injection, subcutaneous injection, intravenous injection, oral        administering, or intranasal administering.    -   19. A method treating or alleviating symptoms of ZIKV in a        subject, comprising the step of administering to the subject the        immunogenic composition of claim 4.    -   20. A method treating or alleviating symptoms of ZIKV in a        subject, comprising the step of administering to the subject the        purified, inactivated immunogenic composition of any of claim 5        or 8.    -   21. A medicament comprising the immunogenic composition of claim        4.    -   22. A medicament comprising the purified, inactivated        immunogenic composition of any of claim 5 or 8.    -   23. A medicament comprising the vaccine any of claim 10 or 11.    -   24. A method of generating a purified inactivated ZIKV        comprising the steps of:        -   i) inoculating a cell culture with an amount of a ZIKV            strain;        -   ii) growing the inoculated virus in cell culture;        -   iii) harvesting and isolating virus fluids from the            inoculated cell culture to prepare a Zika virus concentrate;        -   iv) purifying the ZIKV concentrate;        -   v) inactivating the purified ZIKV; and        -   vi) recovering the purified, inactivated ZIKV.    -   25. A method of generating a purified, inactivated, immunogenic        Zika virus (ZIKV) comprising the steps of:        -   i) inoculating a cell culture with an amount of a ZIKV            strain;        -   ii) growing the inoculated virus in cell culture;        -   iii) harvesting and isolating virus fluids from the            inoculated cell culture to prepare a Zika virus concentrate;        -   iv) purifying the ZIKV concentrate;        -   v) inactivating the purified ZIKV; and        -   vi) recovering the purified, inactivated, and immunogenic            ZIKV.    -   26. The method of claim 24, wherein the purified ZIKV is        inactivated by contacting the ZIKV with a chemical inactivating        agent.    -   27. The method of claim 26, wherein the chemical inactivating        agent is formalin, beta-propiolactone or hydrogen peroxide.    -   28. The method of claim 25, wherein the ZIKV strain used in        step (i) has been passaged at least 3 times in a host cell line.    -   29. The method of claim 26, wherein the ZIKV strain is Puerto        Rico PRVABC59, Thailand SV0127/14, Philippine COC C 0740, or        Brazil Fortaleza/2015, or other suitable strains.    -   30. The method of claim 28, wherein after passaging 2 times the        ZIKV is rederived by RNA transfection in a third passage.    -   31. A purified inactivated ZIKV produced by the method of claim        25.    -   32. The use of a purified, inactivated, immunogenic ZIKV of        claim 2 for the manufacture of a medicament for the prevention        of a Zika virus infection in a host or for the prophylaxis of a        Zika infection in a host believed to have been exposed to a Zika        virus.    -   33. A method treating or alleviating symptoms of ZIKV in a        subject, comprising the step of administering to the subject the        immunogenic composition of any of claims 4 to 9.    -   34. A method of generating a purified inactivated immunogenic        Zika virus (ZIKV) comprising the steps of:        -   inoculating a cell culture with an amount of a ZIKV strain,            wherein the Zika strain is Puerto Rico PRVABC59, Thailand            SV0127/14, Philippine COC C 0740, or Brazil Fortaleza/2015,            or other suitable strains.    -   culturing the inoculated virus in cell culture;        -   harvesting and isolating viral fluids from the inoculated            cell culture to prepare a ZIKV concentrate;        -   purifying the ZIKV concentrate;        -   inactivating the purified ZIKV concentrate producing a            purified, inactivated immunogenic ZIKV concentrate; and        -   recovering the purified, inactivated immunogenic ZIKV            concentrate.    -   35. The method of claim 34, wherein the purified ZIKV is        inactivated by contacting the ZIKV with 0.05% formalin at 22° C.        until complete inactivation can be demonstrated.    -   36. The method of claim 35, wherein the purified ZIKV is        inactivated for about 6 to about 7 days.    -   37. The method of claim 25, wherein the ZIKV strain used in        step (i) is a low passage of less than 10 passages in a host        cell line.    -   38. The method of claim 34, wherein the ZIKV is passaged one to        two times and then the ZIKV is rederived by RNA transfection of        an uninfected cell culture in a third passage.    -   39. A purified inactivated immunogenic ZIKV vaccine produced by        any of the methods of claims 34-38.

Other aspects will be apparent to one of skill in the art upon review ofthe description and exemplary depictions that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the disclosure, there are depicted inthe drawings certain features of the aspects and embodiments of thedisclosure. However, the disclosure is not limited to the precisearrangements and instrumentalities of the aspects depicted in thedrawings.

FIG. 1 provides a generalized process flow chart for one method ofproduction of purified inactivated ZIKV vaccine.

FIG. 2 shows Flow Chart 2 that describes passage 1 of the ZIKV humanisolate in Vero cell cultures.

FIG. 3 shows Flow Chart 3 that describes passage of ZIKV in Vero cellcultures using ZIKV passage 1 as inoculum. This passage is also called“pre-master seed”.

FIG. 4 shows Flow Chart 4 for production of Master Seed passage 3 inVero cell cultures.

FIG. 5 shows Flow Chart 5, passage 4 in Vero cell cultures forproduction of a vaccine lot (drug substance).

FIG. 6 shows Flow Chart 6 vialing of drug product after adsorption toalum.

FIG. 7 shows the results of testing of the purified, inactivated ZIKVvaccine using PRVABC59 as the vaccine strain, in mice. Balb/c mice(N=5/group) received a single immunization by the intramuscular (IM) orsubcutaneous (SQ, also s.c.) routes with 1 μg PIV vaccine with alum oralum alone and were challenged at week 4 by intravenous injection with10⁵ viral particles (VP)(10² plaque-forming units (PFU)) of ZIKV-BR(Brazilian isolate). In FIG. 7a , humoral immune responses were assayedat week 3 following vaccination by Env-specific ELISA. In FIG. 7b ,correlates of protective efficacy are shown. Gray bars reflect medians.P-value show statistical significance by t-tests. The sham in thisexperiment is alum. Each dot represents a single mouse, and the grayline is the mean value calculated among all dots. In FIG. 7a , the Xaxis is the vaccine or sham delivered IM or SQ, and PIV delivered IM orSQ. The Y axis shows the log titer of antibodies against the ZIKV Envgene determined by ELISA. In FIG. 7b , the X axis is the protected (noviremia) versus not protected (measurable viremia) after challenge withZIKV. The Y axis is the same as FIG. 7a . This is significant data,because it shows that the sham does not induce an antibody responsewhile the PIV does induce an antibody response. It also shows that thePIV given by the IM route is superior to the PIV given by the SQ route.Anti-ZIKV antibody titers were correlated with protection followingchallenge.

FIG. 8 shows additional results of testing of the purified, inactivatedZIKV vaccine in mice. FIG. 8 shows the results of testing a vaccineprepared by the method described herein, using PRVABC59 as the vaccinestrain, in mice. The terms are the same as used in FIGS. 7a and 7b . Inthis figure, serum viral loads are shown following ZIKV-BR challenge.The X axis is the number of days after challenge, and the Y axis is thequantity of ZIKV expressed as copies of viral particles per ml of serumtested. The X and Y axis together detail the kinetics of viralreplication following challenge. Each graph in FIG. 8 represents adifferent treatment group. Alum alone is used as the sham compared toZIKV PIV administered in the muscle or subcutaneously. These results aresignificant, because they show that the PIV induces antibody production,and that the antibodies protect against ZIKV (i.e., induce protectiveimmunity). The results for the protected mice versus the non-protectedmice are statistically significant. With the sham there is almostcompletely unrestricted viral replication in the mice while in the ZIKVPIV group viremia is 100% prevented in the IM recipients, and 3 out of 5are 100% protected in the SQ group (and the 2 which had viremia hadsignificantly reduced viremia in terms of quantity and duration ofviremia). Taken together, the results shown in FIGS. 7a-b and 8 showthat the ZIKV PIV induces antibodies in mice, and that these antibodiesprevent viral replication after challenge—even after challenge with adifferent strain of ZKIV than used to make the vaccine.

FIG. 9, Panels A-D show the immunogenicity of the ZIKV PIV vaccine innon-human primates. (FIG. 9A) Env-specific ELISA titers and (FIG. 9B)ZIKV-specific microneutralization (MN50) titers following immunizationof rhesus monkeys by the SQ route with 5 μg ZIKV PIV vaccine at weeks 0and 4 (gray arrows). The maximum measurable log MN50 titer in this assaywas 3.86. Cellular immune responses by IFN-γ ELISPOT assays to prM, Env,Cap, and NS1 at (FIG. 9C) week 2 and (FIG. 9D) week 6. Gray bars reflectmedians.

FIG. 10, Panels A-E show protective efficacy of the ZIKV PIV vaccine innon-human primates. PIV vaccinated and sham control rhesus monkeys(N=8/group) were challenged 4 weeks after immunization with 2 doses ofZIKV PiV, by the SQ route using 10⁶ VP (10³ PFU) of ZIKV-BR or ZIKV-PR.Each group contained 6 female and 2 male animals. Viral loads are shownin (FIG. 10A) plasma, (FIG. 10B) urine, (FIG. 10C) CSF, (FIG. 10D)colorectal secretions, and (FIG. 10E) cervicovaginal secretions. Viralloads were determined on days 0, 1, 2, 3, 4, 5, 6, 7 for the plasmasamples (FIG. 10A) and on days 0, 3, 7 for the other samples (FIG.10B-E). Data is shown for all 8 animals in each panel, except for the 6females for cervicovaginal secretions in (FIG. 10E). P-value showsstatistical significance by Fisher's exact test.

FIG. 11, Panels A-E show data from adoptive transfer studies in mice.(FIG. 11A) Env-specific serum ELISA titers and (FIG. 11B) ZIKV-specificmicroneutralization (MN50) titers in serum from recipient Balb/c mice(N=5/group) 1 hour following adoptive transfer of 5-fold serialdilutions (Groups I, II, III, IV) of IgG purified from PIV vaccinatedrhesus monkeys or sham controls. (FIG. 11C) shows plasma viral loads inmice following challenge with 10⁵ VP (10² PFU) ZIKV-BR. (FIG. 11D, E)Immune correlates of protection. Gray bars are medians. P-valuesindicate statistical significance by t-tests.

FIG. 12, Panels A-B show adoptive transfer studies in rhesus monkeys.(FIG. 12A) ZIKV-specific microneutralization (MN50) titers in serum fromrecipient rhesus monkeys (N=2/group) 1 hour following adoptive transferof 5-fold dilutions (Groups I, II) of IgG purified from PIV-vaccinatedrhesus monkeys or sham controls. (FIG. 12B) Plasma viral loads in rhesusmonkeys following challenge with 10⁶ VP (10³ PFU) ZIKV-BR. Gray barsshow medians.

FIG. 13 shows PIV vaccine schedules for the non-human primates.Immunization and challenge schedules for the ZIKV purified inactivatedvirus (PIV) vaccine. Gray arrows indicate vaccinations, and black arrowsindicate ZIKV challenges. The numbers reflect study weeks. Notably, thisschedule is contemplated for human use as well. The data generated,along with what is already known about PIV for flaviviruses, isreasonably demonstrative of what will be safe and effective in humans.

FIG. 14 shows MN50 titers in the sham controls in the ZIKV PIV vaccinestudy for non-human primates. ZIKV-specific microneutralization (MN50)titers following immunization of rhesus monkeys with sham (alum only) atweeks 0 and 4 (gray arrows). Gray bars reflect medians.

FIG. 15 shows correlation of binding and neutralizing antibody titers inthe ZIKV PIV vaccine study for non-human primates. Correlations ofbinding ELISA titers and microneutralization (MN50) titers at weeks 2and 6 are combined from the ZIKV PIV vaccine study. P-value reflects aSpearman rank-correlation test.

FIG. 16 shows IFN-γ ELISPOT assays in the sham controls in the ZIKV PIVvaccine study in non-human primates. Cellular immune responses aremeasured by IFN-γ ELISPOT assays to prM, Env, Cap, and NS1 at week 2 andweek 6 following immunization of rhesus monkeys. Gray bars reflectmedians.

FIG. 17 show MN50 titers following ZIKV challenge in the ZIKV PIVvaccine study for non-human primates. ZIKV-specific microneutralization(MN50) titers following ZIKV-BR challenge in rhesus monkeys thatreceived the ZIKV PIV vaccine or sham (alum only). The maximummeasurable log MN50 titer in this assay was 3.86. Gray bars are medians.

FIG. 18 shows viral loads in the ZIKV PIV vaccine study in non-humanprimates. Plasma viral loads in PIV vaccinated monkeys and sham controlsfollowing challenge with ZIKV-BR or ZIKV-PR (N=4/group).

FIG. 19 shows results of a human clinical trial of ZIKV PIV. The barsrepresent geometric mean neutralizing (MN50) titers and 95% confidenceintervals for volunteers following one and two doses of ZIKV PIV.

FIG. 20 depicts the geometric mean antibody titers determined by MN 50assay based on the trial study site in human volunteers.

FIG. 21 depicts the reverse cumulative curve of neutralizing antibodytiters depicting the study site and the study day on which the titer wasmeasured. The curves are informative, because they provide acomprehensive look at the antibody titers for the entire cohort at apoint in time. For example, at day 57 more than 40% of the cohort (yaxis) have a titer above 100 (x axis). Approximately 10% of the cohorthas titers of 1000 at this same time point. It is evident that althoughthe Trial Study Site 2 and Trial Study Site 1 have very similar curves;trial study site 3 appears to be an outlier with a higher percentage ofthe cohort having higher titers.

DETAILED DESCRIPTION

Before continuing to describe various aspects and embodiments in furtherdetail, it is to be understood that this disclosure is not limited tospecific compositions or process steps, as such may vary. It must benoted that, as used in this specification and the appended claims, thesingular form “a”, “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art. For example,the CONCISE DICTIONARY OF BIOMEDICINE AND MOLECULAR BIOLOGY, Juo,Pei-Show, 2nd ed., 2002, CRC Press; THE DICTIONARY OF CELL AND MOLECULARBIOLOGY, 3rd ed., 1999, Academic Press; and the OXFORD DICTIONARY OFBIOCHEMISTRY AND MOLECULAR BIOLOGY, Revised, 2000, Oxford UniversityPress, provide one of skill with a general dictionary of many of theterms used herein.

A “vaccine” as referred herein is defined as a pharmaceutical ortherapeutic composition used to inoculate an animal in order to immunizethe animal against infection by an organism, such as ZIKV. Vaccinestypically comprise one or more antigens derived from one or moreorganisms (ZIKV) which on administration to an animal will stimulateactive immunity and protect that animal against infection with these orrelated pathogenic organisms.

By “viruses” is meant different strains (genotypes) of the ZIKV virus,causing the same disease or responsible for different diseases. It isunderstood that the vaccines can combine different strains of ZIKVviruses.

The term “pharmaceutically (or pharmacologically) acceptable” means thatits administration can be tolerated by a recipient patient or subject.An agent is physiologically significant if its presence results in adetectable change in the physiology of a recipient patient. Thecompounds herein can be formulated according to known methods to preparepharmaceutically useful compositions, whereby these materials, or theirfunctional derivatives, are combined in an admixture with apharmaceutically acceptable adjuvant vehicle. Suitable vehicles andtheir formulation, inclusive of other human proteins, e.g., human serumalbumin, are described, for example, in REMINGTON'S PHARMACEUTICALSCIENCES (16th ed., Osol, A. ed., Mack Easton Pa. (1980)). In order toform a pharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofthe above-described compounds together with a suitable amount ofadjuvant.

By “purified” virus, it is meant ZIKV viral particles separated fromhost cell proteins and DNA.

By “adventitious agent, it is meant a contaminant that enters thepassage or production stream beginning with the isolate at the start ofthe process.

The term “consisting essentially of” is intended to encompass thespecified materials, compositions and methods herein and those that donot materially affect the basic and novel characteristic(s) of thematerials, compositions and methods. Basic and novel characteristics ofthe compositions and methods include the purified, inactivated ZIKV, asproduced by the process described herein, and derived from the ZIKVstrains designated as the Puerto Rican strain and the Thai strain. Anexemplary strain is designated PRVABC59 (or the Puerto Rican strain, orZIKV-PR). It was obtained from an outbreak in the Americas, which hadsignificant levels of resulting microcephaly and Guillain-Barresyndrome, so had particular need for a vaccine. In addition, this strainhad a passage history that is acceptable for vaccine development, andhad good early success in yield studies in laboratories. However, themethods for producing and using the PIV described herein would beapplicable for any known strain of ZIKV. The strains are sufficientlyhomologous that the vaccine would be made substantially the same way,and used substantially the same way, for any ZIKV strain. The basic andnovel characteristics also include the use of the purified, inactivatedZIKV as a vaccine or in an immunogenic composition, and all otherrelated uses as described herein. The use of the purified, inactivatedZIKV is effective for the purposes described herein, alone or in thepresence of other ingredients or components. Thus, the presence of otheringredients or components does not materially affect the basic and novelcharacteristics of the compositions described or methods of making andusing the compositions described herein. When used in connection withour novel methods of use, the phrase “consisting essentially of” is amodifier of method steps, such as to include only steps which do notmaterially affect the basic and novel characteristics of the claimedmethod.

The inventors have developed a purified inactivated vaccine (PIV) thatis effective in immunizing a subject against ZIKV infection, and/orpreventing disease and clinical symptoms associated with or caused byZIKV infection. Immunogenic compositions and vaccines comprising theinactivated virus can provide for a global vaccine protecting therecipient from disease caused by any ZIKV strain from any part of theworld—including but not limited to the Puerto Rican strain, the Thailandstrain, the Philippine strain and the Brazilian strain, as well as anystrains circulating in the Americas, Africa and Asia. These strains aregenerally accessible, and most of the sequences of these strains havebeen published. The Asian and America strains are >99% homologous basedon currently available data. Also, our mouse experiment detailed hereinalso supports that the strains are quite homologous—as shown, the mousechallenged with a Brazilian strain was 100% protected by the ZIKVvaccine made with Puerto Rican strain. The non-human primate studiesdescribed below show that rhesus monkeys were 100% protected by the ZIKVvaccine. The mouse and non-human primate data track each othercompletely and yield the same conclusions: anti-Zika antibodies protectagainst infection in mice and non-human primates. ZIKV PIV generatesrobust anti-Zika antibodies in both models. Based on these models, andwhat is already known in the field of flavivirus PIVs, extrapolation tohuman use is reasonable for safe and effective dosages (e.g., a singledose, or a booster dose at 4 weeks as described below).

Other purified inactivated viruses have been successfully employed asvaccines against other viral agents including, for example, Japaneseencephalitis (JE), and tick borne encephalitis, and have been shownexperimentally to have promising results in other diseases such asdengue (DENV) and yellow fever (YFV). As is well understood by personsworking in this field, each of these pathogens has a distinctiveclinical disease associated with it. Zika is unique in that there arecongenital and neurologic outcomes believed to result from autoimmuneversus direct viral effects, and additionally, a purified inactivatedwhole virus is advantageous because of its potential for a superiorsafety profile in special populations like pregnant women. In addition,regarding DENV, the inactivation kinetics (rate of inactivation) aredifferent—only about 1 day is needed to inactivate ZIKV, whereas 2 daysare needed for DENV.

A purified, inactivated vaccine (PIV) provides many advantages relativeto other types of immunogenic products, and particularly attenuated,live viruses. Such advantages of a PIV include an additional margin ofsafety by virtue of the absence of genetic reversion to a virulent, wildtype virus, potentially lower acute reactogenicity followingvaccination, rapid immunization timelines, potential to co-administerwith other vaccines, and the like. Thus, a vaccine comprising apurified, inactivated ZIKV such as described herein can have theadvantages of (1) an excellent safety profile with no risk for reversionand (2) the potential to confer protective immunity more quickly thanlive attenuated vaccines without their undesirable side effects. Notonly are inactivated vaccines more stable and safer than live vaccines,they are usually easier to store and transport as they do not requirerefrigeration. Further such compositions can be easily stored andtransported in a freeze-dried form, which provides for greateraccessibility to people in developing countries.

The vaccines and immunogenic compositions of ZIKV can be made using thefollowing novel method. A live ZIKV is purified so as to remove allpathogens and adventitious agents. The ZIKV can be purified so that noother impurities are present in the final product which could compromisethe safety of the vaccine or immunogenic composition, or interferesignificantly with the immunologic effect and subsequent protectiveoutcome. The ZIKV strain also can be rederived by RNA transfection in adesired passage (e.g., p-3) so that the possibility of adventitiousagents and other contamination is reduced. The product of rederivationis further purified to eliminate host cell protein and DNA. The purifiedZIKV (still technically having infective properties) is then inactivatedso that it is not capable of infecting a host with ZIKV but still hassufficient viral antigenicity and immunogenicity to induce animmunogenic response in a host and/or generate an antibody responsereactive to ZIKV—not infective but strongly immunogenic. For the vaccineembodiments, the purified, inactivated ZIKV is capable of inducing aprotective response.

Any ZIKV strain or isolate or derivative may be used. Examples of ZIKVstrains include the isolates known as Thailand SV0127/14, Philippine COCC 0740, Brazil Fortaleza/2015, and Puerto Rico PRVABC59, although moreare known. Complete genome sequences of the Zika virus strains isolatedfrom the blood of patients in Thailand in 2014 and in the Philippines in2012 are provided in for example the article and associates documents ofEllison et al., “Completed Genome Sequences of Zika Virus StrainsIsolated from the Blood of Patients in Thailand in 2014 and thePhilippines in 2012,” Genome Announcements 4(3): e00359-16. All ZIKVstrains would be useful in the compositions described herein, in all ofits embodiments. The ZIKV strain may be live or attenuated as a startingmaterial. An exemplary strain is the Puerto Rican strain PRVABC59. Wheninactivated, the ZIKV strains are effective in immunogenic compositionsand vaccines. PRVABC59 demonstrated good potential as a vaccine, and asdescribed in FIGS. 2-5, PRVABC59 was used to prepare an effectivevaccine. FIGS. 7 and 8 show the results of successful tests of thisvaccine in mice. In particular, a potential mechanistic correlate ofprotection is shown in the successful use of the Puerto Rican strainvaccine to protect a mouse challenged with the Brazilian strain.Japanese encephalitis, yellow fever, and tick borne encephalitisvaccines have also demonstrated validated correlates of protection whichare antibody-based (ELISA or neutralizing). FIGS. 9-19 show the resultsof successful tests of this vaccine in rhesus monkeys. Immunogenicityand protective efficacy of the PIV is demonstrated. All PIV vaccinatedanimals showed complete protection against ZIKV challenge.

Purification of the ZIKV may be performed by physical or chemicaltechniques or any combinations thereof that are routinely used in theart. Physical methods utilize the physical properties of the virus suchas density, size, mass, sedimentation coefficient, and the like, andinclude but are not limited to, ultracentrifugation, density gradientcentrifugation, ultrafiltration, size-exclusion chromatography, and thelike. Chemical purification can employ methods such asadsorption/desorption through chemical or physiochemical reactions suchas ion exchange chromatography, affinity chromatography, hydrophobicinteraction chromatography, hydroxyapatite matrix, precipitation withinorganic salts such as ammonium sulfate, and the like.

Inactivation of the ZIKV can be done by any method generally known inthe art, as long as the end product is a non-infectious Zika virus thatretains high immunogenicity and preserves viral antigenicity. Forinstance, the ZIKV may be rendered non-infectious bykilling/inactivating the virus by heat, gamma irradiation, UV light, orby contact with a chemical agent, such as formalin or beta-propiolactone(BPL), glutaraldehyde, N-acetylethyleneimine, binary ethyleneimine,tertiary ethyleneimine, ascorbic acid, caprylic acid, psolarens,detergents including non-ionic detergents, and the like. The chemicalinactivating agent is added to a virus suspension in an amount effectiveto inactivate the virus, under conditions that retain highimmunogenicity of the vaccine preparation. An inactivation temperaturecan be 22° C.

For example, inactivation with formalin can be performed at 4-22° C. fora time sufficient to achieve complete inactivation of infectivity whilethe virus particles maintain a protective response (remain immunogenicwhen administered to a host animal), considering also the recommendedthree-fold safety margin since formalin inactivation is non-linear.Inactivation can be performed for 2 or more days, but generally lessthan 10 days. For example, inactivation with formalin can be performedfor about 7 days at 22° C. Optional filtration through a 0.22 μm filtermay be performed, and the filtered material transferred to a freshcontainer at 48 hrs to remove virus aggregates resistant toinactivation. In some embodiments, BPL, which may be faster and exhibitmore linear kinetics, may be used for inactivation. Typically, theinactivating agent is neutralized (e.g., with sodium bisulfite in thecase of formalin) or removed by diafiltration.

The purification—inactivation method for ZIKV can include at least thefollowing steps:

(i) inoculating a cell culture with a ZIKV strain (e.g., a Master Seedor a strain that has been passaged at least 3 times);(ii) propagating the virus in the inoculated cell culture;(iii) rederivation of the strain the transfection to make a Master Seed;(iv) preparing a vaccine lot by inoculation with the Master Seed, andharvesting and isolating

virus fluids from the inoculated cell culture to prepare a ZIKVconcentrate;

(v) treating clarified ZIKV harvest with enzymes or other chemicals thatdegrade host cell DNA to acceptable levels;(vi) concentrating ZIKV virus using an ultrafilter;(vii) purifying the ZIKV concentrate to remove host cell contaminants;(viii) inactivating the purified ZIKV; and(ix) recovering the inactivated purified ZIKV.

Rederivation of the ZIKV—especially for the vaccine Master Seed—by RNAtransfection can be an important step, because this helps provide for acomposition/vaccine that is free from any contaminating adventitiousagents that may otherwise induce an adverse event or side effect whenadministered to a subject, and also provides an additional margin ofsafety. Besides ensuring purity, it allows for absolute traceability ofthe viral strain. At the end of a second passage, RNA can be extracted,and in after a third passage the RNA is used for transfection. Thepassaged ZIKV strain may be re-derived by RNA transfection using anystandard method known in the art, in a suitable cell line such as, forexample, Vero cells that have been certified for vaccine production. There-derived virus may be used to produce a vaccine master seed lot and/ora working seed lot. By “master seed” is meant a seed lot that can beused for vaccine lot production, and it helps ensure the reproducibilityof vaccine lot production.

The purified, inactivated ZIKV is useful to prepare compositions, suchas vaccines, that are effective to generate a prophylactic immuneresponse against ZIKV infection. The compositions, including vaccines,may also (or alternatively) be effective to generate a therapeuticimmune response against ZIKV infection. For example, ZIKV is propagatedto high titers in cell lines suitable for making human-use products.Specifically, ZIKV is passaged and replication-optimized (e.g., in Veromonkey kidney cells), then purified using column chromatography or otherpurification methods and inactivated with, for example, formalin. Thefinal PIV is adjuvanted with alum or other adjuvants that enhanceimmunogenicity. Animals including mice and non-human primates thatreceive injections of the PIV will mount antibody responses that areprotective. If desired, an immune response may be induced in a virusnaïve subject.

As an example of the method of producing the purified, inactivated ZIKV,the following protocol was used. The starting material was a strain ofZIKV adapted to grow in Vero cells by 2-3 cell serial passages at a lowmultiplicity of infection (MOI). Multiple strains of ZIKV were screenedwith the most infectious being selected for development. Specifically,the ZIKV isolates that were tested include Thailand SV0127/14,Philippine COC C 0740, Brazil Fortaleza/2015, and Puerto Rico PRVABC59.

The higher-yielding strains are preferred. For instance, the preferredminimum yield in order to be further down-selected is 7 logs output,after the respective strain is transfected into the host cell. However,as is well known in the art, there are procedures to increase yield sothis list is not exhaustive of the ZIKV strains that can be used toproduce the compositions and vaccines described herein.

The strain selected was the Puerto Rico strain, PRVABC59. The PuertoRico strain initially showed the best yield.

As an example of screening ZIKV isolates for use in ourpurification-inactivation process, the following passaging protocol canbe used. Monolayers of Vero-PM cells (<p-146) can be prepared in 25 cm²flasks. The isolate can be thawed and diluted to an MOI of 0.01 in EMEMdiluent (if titer is known). If titer is not known, a 1:100 dilution forinoculation can be made. Flasks of 2×25 cm² can be inoculated using 1.0mL inoc; let adsorb for 1 h at 35° C.; then 7.0 mL EMEM maintenancemedium can be added to each flask (Safe Operating Procedure SOPM-093-xx). Cytopathic effects (CPE) are daily observed and recorded.From each flask, 0.5 mL is removed daily until CPE has progressed to3-4+. A sample can be added to an equal volume of fetal bovine serum(FBS) and frozen at −80° C. All samples taken can be assayed on Vero-WHOcells using a standard SOP for flavivirus plaque assay (SOP QC-145-xx).

In the method of ZIKV purification and inactivation, the selected ZIKVstrain(s) was re-derived by RNA transfection using standard methods in asuitable cell line (e.g., Vero cells that have been certified forvaccine production) so as to eliminate potential adventitious agents.The re-derived virus was used to produce vaccine master seed lots. Forvaccine lot manufacture the certified Vero cells grown in rollerbottles, cell factories, or suspension cultures were infected with theZIKV master seed at a suitable MOI (e.g., 0.1 to 0.001). After infectionthe cell culture fluids containing the virus were harvested based on thedevelopment of cytopathology (e.g., 50% or more cells showing cytopathiceffects, CPE) and/or viral antigen yields measured by a suitable assaysuch as virus hemagglutination or ELISA. Depending upon the infectiontime course and the amount of cytopathology the virus can also beharvested continuously or at intervals throughout the infection cyclewith replacement of removed culture medium. The collected bulksupernatant harvests were pooled and concentrated approximately 10 to20-fold by a suitable method, (e.g., tangential flow ultrafiltrationusing an appropriate membrane pore size to retain the virus and removesmall MW contaminants). The virus concentrate was then subjected toBenzonase® (Millipore Sigma) treatment or protamine sulfateprecipitation to remove residual host cell nucleic acids andcontaminating cellular proteins. The concentrated, treated virus poolwas then purified by a suitable method such as density gradientcentrifugation, rate zonal centrifugation, continuous flowcentrifugation, or column chromatographically, and the virus peakfractions were identified by HA or ELISA or optical density, and pooled.The purified virus concentrate was quantified for protein, infectivityand viral and host cell antigen content and host nucleic acids.

Inactivation of the purified virus was performed by a suitable methodthat preserves viral antigenicity such as formalin orbeta-propriolactone (BPL). For example, inactivation with formalin canbe performed at 4° C. to 22° C. for a time sufficient to achievecomplete inactivation of infectivity, considering also the recommendedthree-fold safety margin since formalin inactivation is non-linear, withfiltration through a 0.22 μm filter and transfer to a fresh container at48 hrs to remove virus aggregates resistant to inactivation. Similarly,BPL, which is faster and exhibits more linear kinetics, can also beused. The inactivating agent is typically neutralized (e.g., with sodiumbisulfite in the case of formalin) or removed by diafiltration.

Bulk vaccines can be tested for sterility, protein, antigen and nucleicacid content using established assays. Residual infectivity can beassayed by inoculation of approximately 5% of the lot volume onto Verocell cultures, or another suitable cell line, followed by incubation fora sufficient time to amplify any residual infectious virus present,which can then be detected by IFA directly on the cells or by a plaqueassay of the culture supernatants. Following inactivation the bulkvaccines can be mixed with suitable excipients and/or stabilizers andstored frozen (e.g., −20° C. to −80° C. prior to formulation).Inactivated ZIKV bulk can be diluted to a protein concentration thatwill be suitable for a human immunizing dose. Final, vialed vaccine canbe tested for purity, identity, osmolality, endotoxin, and sterility byvarious, standardized assays.

The method can be useful to produce a purified, inactivated ZIKV thatmay be used for production of vaccine lots. The method entails infectionof a suitable cell line for vaccine manufacture—for example, certifiedVero cells grown in roller bottles, cell factories, or suspensioncultures can be infected with the ZIKV master seed at a suitable MOI(e.g., 0.1 to 0.001). By “master seed” is meant the seed that issuitable for use for multiple lots of vaccine lot production, it can bethe result of our process that includes RNA transfection. It isthoroughly tested for adventitious agents and other contaminants. Afterinfection the cell culture fluids containing the virus can be harvestedbased on the development of cytopathology (e.g., 50% or more cellsshowing cytopathic effects, CPE) and/or viral antigen yields measured bya suitable assay such as virus hemagglutination (HA) or ELISA. Dependingupon the infection time course and the amount of cytopathology the virusmay also be harvested continuously or at intervals throughout theinfection cycle with replacement of removed culture medium. Thecollected bulk supernatant harvests can be pooled and concentratedapproximately 10 to 20-dfold by a suitable method, (e.g., tangentialflow ultrafiltration using an appropriate membrane pore size to retainthe virus and remove small MW contaminants). The virus concentrate canbe subjected to a treatment that removes residual host cell nucleicacids and contaminating cellular proteins such as, for example,Benzonase® treatment or protamine sulfate precipitation. Theconcentrated, treated virus pool may then be purified by a suitablemethod such as density gradient centrifugation, rate zonalcentrifugation, continuous flow centrifugation, or columnchromatographically, and the virus peak fractions may be identified byoptical density (OD), HA or ELISA, and pooled. The purified virusconcentrate can be quantified for protein, infectivity and viral andhost cell antigen content and host nucleic acids.

Bulk vaccines may be tested for sterility, protein, antigen and nucleicacid content using established assays. Residual infectivity can beassayed by inoculation of approximately 5% of the lot volume onto Verocell cultures, or another suitable cell line, followed by incubation fora sufficient time to amplify any residual infectious virus present,which can then be detected by IFA directly on the cells or by plaqueassay of the culture supernatants. Following inactivation the bulkvaccines can be mixed with suitable excipients and/or stabilizers andstored frozen (e.g., −20° C. to −80° C. prior to formulation).Inactivated ZIKV bulk may be diluted to a protein concentration that issuitable for an immunizing dose in a subject (e.g., a mammal such as ahuman). The final, vialed vaccine may be tested for purity, identity,osmolality, endotoxin, and sterility by various, standardized assaysgenerally known in the art.

Immunogenic potency of bulk vaccine lots and the final formulation canbe tested by administering the vaccines to mice. Typically, groups often 5-6 week-old, female, Swiss-ICR mice receive serially graded dosesranging from about one nanogram to one microgram of vaccine, as requiredto reach an endpoint, in a 0.2 ml intramuscular or subcutaneous dose. Acorresponding control group receives saline or saline plus adjuvant, asappropriate. Mice are typically boosted once; this can be done on day 14or 28 after priming, and then blood is collected one to two weeks later.The sera from individual mice are assayed for virus neutralizingantibodies and the vaccine median immunizing dose (ID50) is calculated.In this way vaccine potency and stability may be monitored periodically.

An animal efficacy study is designed to demonstrate that the vaccine issafe and has the potential for clinical benefit in human populations.Animal models may be infection models or disease models. If the animalexperiences disease similar to humans this is a disease model and isdesired. In the event animals do not experience disease manifestationsafter exposure but viral replication (viremia) is measurable it ispossible to extrapolate animal results to potential outcomes in humans(prevent viremia=prevent disease). Therefore, if vaccination does notcause any adverse events in the animal and induces an effective immuneresponse (neutralizing antibodies) which protects against a live viruschallenge in comparison to a placebo or another control this istypically supportive data to advance to human trials. This testing maybe necessary before a vaccine can progress to a clinical trial.Typically, such experiments are best performed in a non-human primateinfection model (e.g., rhesus macaques) with the primary endpoints beingthe measurement of virus neutralizing antibodies after vaccination andthe measurement of protection against challenge with an attenuated orwild type ZIKV strain. Protection can be assessed by a disease surrogatesuch as circulating virus (viremia) after virus challenge with anear-wild-type (low passage) strain of ZIKV. Various vaccine doses andimmunization schedules can also be tested in the experiment. Group sizesof 5 to 10 are suitable for a pilot study. For example, using Fisher'sExact Test with alpha=0.05 (2-sided) and n=5 animals per group: for 100%vs. 0%, or 100% vs. 5%, the power is about 80%. Responses can becompared and contrasted for individual animals and among groups usingstandard statistical methods. For example, log-transformed antibody andviremia titers can be analyzed by ANOVA. Fisher's exact test can be usedto compare rates of seroconversion to each virus antigen and viremiarates among vaccine groups and placebo controls. A one-way analysis ofvariance with a contrast test for trend may be used to assessdifferences in antibody or viremia titers among groups. To stabilize thevariance the analysis is conducted on the logs of the quantifiedresponses. A test for trend using the logistic model can be used toassess differences in the proportion of seroconverters.

Reactogenicity of the vaccines disclosed herein may be monitored andevaluated as may be necessary. A reactogenicity event is typicallyidentified as an adverse event that is commonly known to occur for thecandidate therapeutic/prophylactic product being studied. Typically,such events are collected in a standard, systematic format using agraded scale based on functional assessment or magnitude of reaction.This helps to provide a risk profile of the candidate product and adefined listing of expected (or unexpected) adverse events, and whethersuch events are local or systemic events.

The vaccines described herein may offer good immune protection againstmultiple (heterologous) strains of ZIKV in addition to the particularZIKV strain(s) used in production of the vaccine. The ZIKV isolates mayexhibit broad neutralizing activity and may cross-neutralize differentgenotypes/genotypic variants/strains of ZIKV. This occurrence wasdemonstrated in the murine study described herein, wherein the PuertoRican vaccine strain protected against challenged by the Brazilianstrain.

The purified and inactivated ZIKV vaccine is prepared for administrationto mammals, suitably humans, mice, rats or rabbits, by methods known inthe art, which can include filtering to sterilize the solution, dilutingthe solution, adding an adjuvant and stabilizing the solution.

The vaccines disclosed herein may be administered to a human or animalby a number of routes, including but not limited to, for example,parenterally (e.g. intramuscularly, transdermally), intranasally,orally, topically, or other routes know by one skilled in the art. Theterm parenteral as used hereinafter includes intravenous, subcutaneous,intradermal, intramuscular, intra-arterial injection, or by infusiontechniques. The vaccine may be in the form of a single dose preparationor in multi-dose vials which can be used for mass vaccination programs.Suitable methods of preparing and using vaccines can be found inREMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co., Easton, Pa.,Osol (ed.) (1980) and New TRENDS IN DEVELOPMENTS IN VACCINES, Voller etal. (eds.), University Park Press, Baltimore, Md. (1978), incorporatedby reference.

In some embodiments, a vaccine composition as disclosed herein may beadministered parenterally in dosage unit formulations containingstandard, well-known nontoxic physiologically acceptable adjuvants,and/or vehicles.

In some embodiments, the vaccine compositions may further comprise oneor more adjuvants. An “adjuvant” is a substance that serves to enhance,accelerate, or prolong the antigen-specific immune response of anantigen when used in combination with specific vaccine antigens but donot stimulate an immune response when used alone. Suitable adjuvantsinclude inorganic or organic adjuvants. Suitable inorganic adjuvantsinclude, but are not limited to, for example, an aluminum salt, such asaluminum hydroxide gel (alum) or aluminum phosphate, but may also be asalt of calcium (particularly calcium carbonate), iron or zinc, or maybe an insoluble suspension of acylated tyrosine, or acylated sugars,cationically or anionically derivitised polysaccharides orpolyphospharenes. Other suitable adjuvants are known to one skilled inthe art. Suitable Th1 adjuvant systems may also be used, and include,but are not limited to, for example, Monophosphphorly lipid A, othernon-toxic derivatives of LPS, and combination of monophosphoryl lipid A,such as 3-de-O-acrylated monophosphorly lipid A (# D-MPL) together withan aluminum salt.

Other suitable examples of adjuvants include, but are not limited to,MF59, MPLA, Mycobacterium tuberculosis, Bordetella pertussis, bacteriallipopolysaccharides, aminoalkyl glucosamine phosphate compounds (AGP),or derivatives or analogs thereof, which are available from Corixa(Hamilton, Mont.), and which are described in U.S. Pat. No. 6,113,918;e.g., 2-[(R)-3-Tetradecanoyloxytetradecanoylamino]ethyl,2-Deoxy-4-O-phosphono-3-O—[(R)-3-tetradecanoyoxytetradecanoy1]-2-[(R)-3-tetradecanoyoxytetradecanoylamino]-b-D-glucopyra noside,MPL™ (3-O-deacylated monophosphoryl lipid A) (available from Corixa)described in U.S. Pat. No. 4,912,094, synthetic polynucleotides such asoligonucleotides containing a CpG motif (U.S. Pat. No. 6,207,646),COG-ODN (CpG oligodeoxynucleotides), polypeptides, saponins such as QuilA or STIMULON™ QS-21 (Antigenics, Framingham, Mass.), described in U.S.Pat. No. 5,057,540, a pertussis toxin (PT), or an E. coli heat-labiletoxin (LT), particularly LT-K63, LT-R72, CT-5109, PT-K9/G129; see, e.g.,International Patent Publication Nos. WO 93/13302 and WO 92/19265,cholera toxin (either in a wild-type or mutant form). Alternatively,various oil formulations such as stearyl tyrosine (ST, see U.S. Pat. No.4,258,029), the dipeptide known as MDP, saponin, cholera toxin B subunit(CTB), a heat labile enterotoxin (LT) from E. coli (a geneticallytoxoided mutant LT has been developed), and Emulsomes (Pharmos, LTD.,Rehovot, Israel). Various cytokines and lymphokines are suitable for useas adjuvants. One such adjuvant is granulocyte-macrophage colonystimulating factor (GM-CSF), which has a nucleotide sequence asdescribed in U.S. Pat. No. 5,078,996. The cytokine interleukin-12(IL-12) is another adjuvant which is described in U.S. Pat. No.5,723,127. Other cytokines or lymphokines have been shown to have immunemodulating activity, including, but not limited to, the interleukins1-alpha (IL-1α), 1-beta (IL-1β), 2 (IL-2), 4 (IL-4), 5 (IL-5), 6 (IL-6),7 (IL-7), 8 (IL-8), 10 (IL-10), 13 (IL-13), 14 (IL-14), 15 (IL-15), 16(IL-16), 17 (IL-17) and 18(IL-18), the interferons-alpha (IFNα), beta(IFN1β) and gamma (IFNγ), granulocyte colony stimulating factor, and thetumor necrosis factors alpha and beta (TNFα and TNFβ respectively), andare suitable for use as adjuvants.

The vaccine compositions can be lyophilized to produce a vaccine againstZIKV in a dried form for ease in transportation and storage. Further,the vaccine may be prepared in the form of a mixed vaccine whichcontains the inactivated virus described herein and at least one otherantigen as long as the added antigen does not interfere with the abilityand/or efficacy of the vaccine, and as long as the added antigen doesnot induce additive or synergistic side effects and/or adversereactions. The vaccine can be associated with chemical moieties whichmay improve the vaccine's solubility, absorption, biological half-life,etc. The moieties may alternatively decrease the toxicity of thevaccine, eliminate or attenuate any undesirable side effect of thevaccine, etc. Moieties capable of mediating such effects are disclosedin REMINGTON'S PHARMACEUTICAL SCIENCES (1980) and later editions.Procedures for coupling such moieties to a molecule are well known inthe art.

The vaccine may be stored in a sealed vial, ampule or the like. Thevaccines disclosed herein can generally be administered in the form of aspray for intranasal administration, or by nose drops, inhalants, swabson tonsils, or a capsule, liquid, suspension or elixirs for oraladministration. In the case where the vaccine is in a dried form, thevaccine is dissolved or suspended in sterilized distilled water beforeadministration.

Vaccine compositions disclosed herein may include an adjuvant. If in asolution or a liquid aerosol suspension, suitable adjuvants can include,but are not limited to, salt solution, sucrose solution, or otherpharmaceutically acceptable buffer solutions. Aerosol solutions mayfurther comprise a surfactant.

Among the acceptable vehicles and solvents that may be used includewater, Ringer's solution, and isotonic sodium chloride solution,including saline solutions buffered with phosphate, lactate, Tris, andthe like. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium, including, but not limited to, forexample, synthetic mono- or di-glycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

Injectable preparations, for example sterile injectable aqueous oroleaginous suspensions, are formulated according to the known art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation are also a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent, forexample, as a solution in 1,3-butanediol.

Some aspects are illustrated by the following examples. These examplesare provided to describe specific embodiments of the technology and donot limit the scope of the disclosure. It will be understood by thoseskilled in the art that the full scope of the disclosure is defined bythe claims appending this specification, and any alterations,modifications, or equivalents of those claims.

EXAMPLES Example 1. Engineering Non-GMP Lot Production

FIG. 2 includes Flow Chart 1 with details of the process used to make apurified and inactivated ZIKV. The ZIKV isolate was PRVABC59. The smallboxes to the right indicate tests done at each step of the process. Theresult of this process was the lot that was ultimately used in the mousestudies described below.

Example 2. ZIKV Purified, Inactivated Vaccine GMP Production withPRVABC59

FIGS. 4-7 include Flow Charts 2-5, with details of how ZIKV isolatePRVBC59 is used to make ZIKV vaccine. The small boxes to the rightindicate tests done at each step of the process. The passage series islimited to 4 (including the final passage, as part of thepurification-inactivation process) to economize time and effort to getthe vaccine made. It proved to be a sufficient minimum passage number.

Example 3. Immunizing Dose Identified in Mouse Potency Assay

Animals.

Balb/c, SJL, and C57BL/6 mice were purchased from Jackson Laboratories(Bar Harbor, Me., USA). Mice are vaccinated with 50 μg DNA vaccines bythe i.m. (intramuscularly) route or with 1 μg PIV vaccines with alumadjuvant by the i.m. or SQ routes and are then challenged by the i.v.route with 10⁵ viral particles (VP) [10² plaque-forming units (PFU)]ZIKV-BR]. Immunologic and virologic assays are performed blinded. Allanimal studies are approved by the BIDMC Institutional Animal Care andUse Committee (IACUC).

PIV Vaccine.

The ZIKV purified inactivated vaccine (PIV) are produced at the PilotBioproduction Facility, Walter Reed Army Institute of Research, SilverSpring, Md., USA. The PIV vaccine is based on the Puerto Rican ZIKVPRVABC59 isolate, which was obtained from the Centers for DiseaseControl and Prevention, Fort Collins, Colo., USA (also available fromAmerican Type Culture Collection at ATCC No. VR-1843, Manassas, Va.20108 USA); the PRVABC59 strain can also be obtained from BEI Resources.The Vero cells used for passage and vaccine production were a derivativeof a certified cell line manufactured at The Salk Institute, Swiftwater,Pa. After inoculation, virus is harvested on day 5. Harvested virus ispooled and clarified by centrifugation followed by filtration andultrafiltration (100,000 molecular weight cutoff). To remove cellularprotein and DNA, concentrated virus is treated with benzonase andpurified using column chromatography. Column fractions are collectedbased on optical density associated with viral particles. Positivefractions are pooled, diluted, and formalin was added at a concentrationof 0.05% (v/v). After 7 days inactivation at 22° C., formalin is removedby dialysis, and the bulk vaccine is stored at 4° C. Testing prior touse confirmed virus inactivation by PFU assays.

ZIKV Challenge Stocks.

ZIKV stocks were provided by University of Sao Paulo, Brazil (BrazilZK2015; ZIKV—BR¹⁰) and the Centers for Disease Control and Prevention,USA (Puerto Rico PRVABC59; ZIKV-PR). Both strains were at passage number3. Low passage number Vero E6 cells were then infected at an MOI of 0.01PFU/cell. Supernatant is screened daily for viral titers and harvestedat peak growth. Culture supernatants are clarified by centrifugation,and fetal bovine serum was added to 20% final concentration (v/v) andstored at −80° C. The concentration and infectivity of the stocks aredetermined by RT-PCR (reverse transcriptase polymerase chain reaction)and PFU assays. The viral particle (VP) to plaque-forming unit (PFU)ratio of both stocks is approximately 1,000.

Using a purified inactivated virus (PIV) vaccine derived from the PuertoRico PRVABC59 strain, groups of Balb/c mice (N=5/group) are administereda single immunization of 1 μg of the PIV vaccine with alum adjuvant oradjuvant alone by the i.m. or s.q. (SQ or s.c.) routes. Antibody titersare substantially higher in the group that received the PIV vaccine bythe i.m. route as compared with the SQ route (FIG. 7a ). At week 4, allmice are challenged with ZIKV-BR by the i.v. route with 10⁵ viralparticles (VP) [10² plaque-forming units (PFU)] of ZIKV-BR.

Complete protection is observed in the group that received the PIVvaccine by the i.m. route (FIG. 7b and FIG. 8). Two mice that receivedthe PIV vaccine by the SQ route show low levels of viremia (FIG. 7),consistent with the lower Env-specific antibody titers in this group(P=0.001, FIG. 7b ). These data demonstrate that vaccine protectionagainst ZIKV-BR can be achieved in this model with the PIV vaccine thatis designed to induce Env-specific antibodies.

The data demonstrate that a single immunization with this PIV vaccineprovides complete protection against parenteral ZIKV challenges in mice.Protective efficacy is mediated by vaccine-elicited Env-specificantibodies, as evidenced by statistical analyses of immune correlates ofprotection (FIG. 7b ).

Example 4. Protective Efficacy of PIV Against Zika Challenge inNon-Human Primates

Having successfully shown protective efficacy of a ZIKV purifiedinactivated vaccine against ZIKV challenges in mice, we evaluated theimmunogenicity and protective efficacy of the PIV in ZIKV challengestudies in rhesus monkeys. Adoptive transfer studies are conducteddemonstrating that the ZIKV PIV created antibodies and these antibodiesprotect against infection.

Materials and Methods

Animals, Vaccines, and Challenges.

Thirty-four (34) outbred, Indian-origin male and female rhesus monkeys(Macaca mulatta) were housed at Bioqual, Rockville, Md. Monkeys areimmunized by the s.c. route with 5 μg ZIKV purified inactivated virus(PIV) vaccine derived from the PRVABC59 isolate (27) with alum(Alhydrogel; Brenntag Biosector, Denmark) or alum alone at weeks 0 and 4(N=8/group). Rhesus monkeys are challenged four weeks after the finalimmunization by the s.c (SQ) route with 10⁶ viral particles (VP) [10³plaque-forming units (PFU)] ZIKV-BR (Brazil ZKV2015) or ZIKV-PR(PRVABC59) (15).

For adoptive transfer studies, Balb/c mice are infused i.v. with IgGpurified from PIV vaccinated monkeys at week 8 and were challenged bythe i.v. route with 10⁵ VP (10² PFU) ZIKV-BR. Rhesus monkeys are infusedi.v. with IgG purified from PIV vaccinated monkeys at week 8 and werechallenged by the s.c. (SQ) route with 10⁶ VP (10³ PFU) ZIKV-BR. Animalswere randomly allocated to groups. Immunologic and virologic assays wereperformed blinded. All animal studies were approved by the appropriateInstitutional Animal Care and Use Committee (IACUC).

RT-PCR.

RT-PCR assays are utilized to monitor viral loads, essentially aspreviously described (27). RNA is extracted from plasma or other sampleswith a QIAcube HT (Qiagen, Germany). The wildtype ZIKV BeH815744 Capgene is utilized as a standard (see GenBank KU365780; Brazil strain;Larocca et al., “Vaccine protection against Zika virus from Brazil,”Nature 536: 474-478 (Aug. 25, 2016). RNA is purified (Zymo Research, CA,USA), and RNA quality and concentration was assessed by the BIDMCMolecular Core Facility. Log dilutions of the RNA standard are reversetranscribed and included with each RT-PCR assay. Viral loads arecalculated as virus particles (VP) per ml and were confirmed by PFUassays. Assay sensitivity was 100 copies/ml.

Pfu Assay.

Vero WHO cells are seeded in a MW6 plate to reach confluency at day 3.Cells are infected with log dilutions of ZIKV for 1 h and overlayed withagar. Cells are stained after 6 days of infection by neutral redstaining. Plaques are counted, and titers are calculated by multiplyingthe number of plaques by the dilution and divided by the infectionvolume.

ELISA.

Monkey ZIKV Env ELISA kits (Alpha Diagnostic International, TX, USA) areused to determine endpoint binding antibody titers using a modifiedprotocol. 96-well plates coated with ZIKV Env protein were firstequilibrated at room temperature with 300 μl of kit working wash bufferfor 5 min. 6 μl of monkey serum is added to the top row, and 3-foldserial dilutions are tested in the remaining rows. Samples are incubatedat room temperature for 1 h, and plates are washed 4 times. 100 μl ofanti-human IgG HRP-conjugate working solution is then added to each welland incubated for 30 min at room temperature. Plates are washed 5 times,developed for 15 min at room temperature with 100 μl of TMB substrate,and the reaction stopped by the addition of 100 μl of stop solution.Plates were analyzed at 450 nm/550 nm on a VersaMax microplate readerusing Softmax Pro 6.0 software (Molecular Devices, CA, USA). ELISAendpoint titers are defined as the highest reciprocal serum dilutionthat yielded an absorbance >2-fold over background values. Log 10endpoint titers are reported.

Neutralization Assay.

A high-throughput ZIKV microneutralization (MN) assay is utilized formeasuring ZIKV-specific neutralizing antibodies, essentially aspreviously described (27). Briefly, serum samples are serially dilutedthree-fold in 96-well micro-plates, and 100 μl of ZIKV-PR containing 100PFU are added to 100 μl of each serum dilution and incubated at 35° C.for 2 h. Supernatants are then transferred to microtiter platescontaining confluent Vero cell monolayers (World Health Organization,NICSC-011038011038). After incubation for 4 d, cells are fixed withabsolute ethanol: methanol for 1 hour at −20° C. and washed three timeswith PBS. The pan-flavivirus monoclonal antibody 6B6-C1 conjugated toHRP (6B6-C1 was a gift from J. T. Roehrig, CDC) is then added to eachwell, incubated at 35° C. for 2 h, and washed with PBS. Plates arewashed, developed with 3,3′,5,5′—tetramethylbenzidine (TMB) for 50 minat room temperature; the reaction is stopped with 1:25 phosphoric acid,and absorbance is read at 450 nm. For a valid assay, the averageabsorbance at 450 nm of three non-infected control wells had to be ≤0.5,and virus-only control wells had to be ≥0.9. Normalized absorbancevalues are calculated, the MN50 titer is determined by a log mid-pointlinear regression model. The MN50 titer is calculated as the reciprocalof the serum dilution that neutralized ≥50% of ZIKV, and seropositivityis defined as a titer ≥10, with the maximum measurable titer 7,290. Log10 MN50 titers are reported.

Antibody Peptide Microarrays.

IgG binding to linear peptides spanning ZIKV Env is measured withpeptide microarrays (JPT Peptide Technologies, Berlin, Germany),essentially as previously described (29). Briefly, microarrays consistedof 3 identical subarrays containing 153 overlapping 15 amino acid ZIKVEnv peptides, which covered 98.2% of available ZIKV Env sequences. Serumis incubated with the microarrays and Alexa Fluor® 647-conjugatedanti-human IgG. The readout and image processing was performed withGenepix 4300A scanner/software. Mean fluorescent intensity (MFI) equaledthe mean of triplicate peptides and is corrected by subtracting valuesfrom matched peptides on control microarrays incubated with secondaryantibody alone. The threshold for positivity was >5× noise distributionof the sample size.

ELISPOT.

ZIKV-specific cellular immune responses are assessed by interferon-γ(IFN-γ) ELISPOT assays using pools of overlapping 15-amino-acid peptidescovering the prM, Env, Cap, and NS1 proteins (JPT, Berlin, Germany),essentially as previously described (27). 96-well multiscreen plates(Millipore, Mass., USA) are coated overnight with 100 μl/well of 10μg/ml anti-human IFN-γ (BD Biosciences, CA, USA) in endotoxin-freeDulbecco's PBS (D-PBS). The plates are then washed three times withD-PBS containing 0.25% Tween-20 (D-PBS-Tween), blocked for 2 h withD-PBS containing 5% FBS at 37° C., washed three times with D-PBS-Tween,rinsed with RPMI 1640 containing 10% FBS to remove the Tween 20, andincubated with 2 μg/ml of each peptide and 2><105 monkey PBMC(peripheral blood mononuclear cells) in triplicate in 100 μl reactionmixture volumes. Following an 18 h incubation at 37° C., the plates arewashed nine times with PBS-Tween and once with distilled water. Theplates are then incubated with 2 μg/ml biotinylated anti-human IFN-γ (BDBiosciences, CA, USA) for 2 h at room temperature, washed six times withPBS-Tween, and incubated for 2 h with a 1:500 dilution ofstreptavidin-alkaline phosphatase (Southern Biotechnology Associates,AL, USA). Following five washes with PBS-Tween and one with PBS, theplates are developed with nitrobluetetrazolium-5-bromo-4-chloro-3-indolyl-phosphate chromogen (Pierce,Ill., USA), stopped by washing with tap water, air dried, and read usingan ELISPOT reader (Cellular Technology Ltd., OH, USA). The numbers ofspot-forming cells (SFC) per 10⁶ cells were calculated. The mediumbackground levels were typically <15 SFC per 10⁶ cells.

IgG Purification and Adoptive Transfer.

Polyclonal IgG was purified from plasma from PIV vaccinated monkeys atweek 8 using protein G purification kits and pooled (Thermo FisherScientific, MA, USA). The purified IgG preparation had a log ELISA titerof 3.30 and a log MN50 titer of 3.30. Purified IgG was infused intogroups of naïve recipient Balb/c mice or rhesus monkeys by 5-fold serialdilutions prior to ZIKV-BR (Zika Brazil strain) challenge. Mice received200, 40, 8, 1.5, or 0 μl of the IgG preparation. Monkeys received 10, 2,or 0 ml of the IgG preparation.

Statistical Analyses.

Analysis of virologic and immunologic data is performed using GraphPadPrism v6.03 (GraphPad Software, CA, USA). Comparisons of groups areperformed using t-tests and Wilcoxon rank-sum tests. Correlations areassessed by Spearman rank-correlation tests.

Vaccine Study in Rhesus Monkeys.

Sixteen rhesus monkeys are immunized by the subcutaneous route with 5 μgZIKV PIV vaccine with alum (N=8) or sham vaccine (alum only) (N=8) atweeks 0 and 4 (FIG. 13). All PIV vaccinated animals developed ZIKVEnv-specific binding antibodies by ELISA as well as ZIKV-specificneutralizing antibodies by microneutralization (MN50) assays at week 2following initial immunization. Median log antibody titers at week 2 are1.87 by ELISA (FIG. 9A) and 2.27 by MN50 assays (FIG. 9B). Following theweek 4 boost immunization, median log antibody titers increasedsubstantially to 3.54 by ELISA (FIG. 9A) and 3.66 by MN50 assays (FIG.9B) at week 6. In contrast, sham control monkeys did not developdetectable ZIKV-specific antibody responses (FIG. 14). Binding antibodytiters correlated with neutralizing antibody titers in the PIVvaccinated animals (P<0.0001, R=0.88, Spearman rank correlation test;FIG. 15), although only minimal antibody-dependent cellular phagocytosisresponses were observed. The majority of PIV vaccinated monkeys (FIGS.9C-D), but not sham control animals (FIG. 16), also developed modestcellular immune responses, primarily to Env, as measured by interferon(IFN)-γ ELISPOT assays.

To assess the protective efficacy of the PIV vaccine against ZIKVchallenge, we infected PIV immunized and sham control monkeys by thesubcutaneous route with 10⁶ viral particles (VP) [10³ plaque-formingunits (PFU)] of ZIKV-BR or ZIKV-PR (N=4/group) (27). Viral loadsfollowing ZIKV challenge are quantitated by RT-PCR (27), and viralinfectivity is confirmed by growth in Vero cells. ZIKV-specific MN50titers increased following challenge, particularly in the sham controls(FIG. 17). Sham control monkeys exhibited 6-7 days of detectable viremiawith median peak viral loads of 5.82 log copies/ml (range 5.21-6.29 logcopies/ml; N=8) on day 3-5 following challenge (FIG. 10A). Virus wasalso detected in the majority of sham control animals in urine andcerebrospinal fluid (CSF) on day 3, as well as in colorectal secretionsand cervicovaginal secretions on day 7 (FIG. 10B-E). In contrast, PIVvaccinated monkeys show complete protection against ZIKV challenge, asevidenced by no detectable virus (<100 copies/ml) in blood, urine, CSF,colorectal secretions, and cervicovaginal secretions in all animalsfollowing challenge (N=8; P=0.0002, Fisher's exact test comparing PIVvaccinated animals vs. sham controls). We were unable to assess ZIKV insemen in the male animals in this study due to inadequate samplevolumes. No major differences in plasma viral loads were observedbetween the sham controls that received ZIKV-BR vs. ZIKV-PR (FIG. 18).

In this study, we demonstrated that our PIV platform provided completeprotection against ZIKV challenge in rhesus monkeys. No specificclinical safety adverse effects related to the vaccine were observed.The protective efficacy of this ZIKV PIV vaccine in mice is describedabove (27). The present data confirm and extend these prior studies bydemonstrating robust protection with these vaccines against ZIKVchallenge in nonhuman primates, and specifically utilizing the dose,route, and schedule of these vaccines that are typically evaluated inclinical trials.

Adoptive Transfer Studies in Mice and Non-Human Primates.

The mechanism of the observed protection by adoptive transfer studieswas explored. IgG is purified from plasma from ZIKV PIV vaccinatedmonkeys at week 8 by protein G affinity chromatography.Vaccine-elicited, ZIKV-specific IgG is then infused into four groups ofnaïve Balb/c mice (N=5/group) by 5-fold serial dilutions of the purifiedIgG preparation, which had a log ELISA titer of 3.30 and a log MN50titer of 3.30. Following infusion, these groups of recipient mice(designated I, II, III, IV) had median log ELISA titers of 2.83, 2.35,1.40, and <1.00 (FIG. 11A) and median log MN50 titers of 2.93, 1.77,1.14, and <1.00 (FIG. 11B). Mice are then challenged by the intravenousroute with 10⁵ VP (10² PFU) of ZIKV-BR, as previously described (27).The higher two doses of purified IgG provides complete protectionfollowing ZIKV challenge, whereas the lower two doses of purified IgGresults in reduced viremia as compared with sham infused control mice(FIG. 11C-E).

Vaccine-elicited, ZIKV-specific IgG was also infused into two groups ofnaïve rhesus monkeys (N=2/group). Following infusion, these groups ofrecipient monkeys (designated I, II) had median log MN50 titers of 2.11and 1.22 (FIG. 12A). Monkeys are then challenged with 10⁶ VP (10³ PFU)of ZIKV-BR. In the animals that received the higher IgG dose, one animalis completely protected and the other showed a blip of viremia on days3-5 (FIG. 12B). No enhancement of viral replication was observed atsubtherapeutic IgG concentrations. Taken together, these datademonstrate that purified IgG from ZIKV PIV vaccinated rhesus monkeysprovided passive protection following adoptive transfer in both rodentsand primates. Therefore, the PIV ZIKV produced from the Puerto Ricanstrain (PRVABC59) is capable of producing ZIKV-specific neutralizingantibodies and completely protected monkeys against ZIKV strains fromboth Brazil and Puerto Rico.

The adoptive transfer studies demonstrate that vaccine-elicitedantibodies are sufficient for protection against ZIKV challenge.Moreover, passive protection in mice and rhesus monkeys is observed atrelatively low antibody titers (FIGS. 11, 12). Such antibody titers arelikely achievable by these vaccine platforms in humans, thus raisingoptimism for the development of a ZIKV vaccine for humans. Notably, thiscan have implication for impact of cross-reactive antibodies againstdengue virus and other flaviviruses. Secondary infection with aheterologous dengue serotype can be clinically more severe than initialinfection, which may or may not reflect antibody-dependent enhancement(30, 31). Cross-reactive antibodies between ZIKV and dengue virus havealso been described (32, 33), and dengue-specific antibodies have beenreported to increase ZIKV replication in vitro (34).

The consistent and robust antibody-based correlates of vaccineprotection against ZIKV challenge in both rodents and primates suggestthe generalizability of these findings. The mouse and primate data trackeach other well, and yield the same conclusions: Zika PIV generatesrobust antibodies to Zika, and those antibodies protect both speciesagainst Zika infection. Similar correlates of protection, andspecifically neutralizing antibody titers >10, have been reported forother flavivirus vaccines in humans (35-37). Taken together, these dataare reasonably predictive for how ZIKV vaccines will perform in humans.PIV vaccines have been evaluated previously in clinical trials for otherflaviviruses, including dengue virus, tick-borne encephalitis virus, andJapanese encephalitis virus (38-42).

The production process described herein is suitable for furtherdevelopment and production of an inactivated ZIKV vaccine.

Comparison of the PIV vaccine for Zika virus with an adenovirus vectorbased vaccine was also performed. The ZIKV pre-membrane (prM) and Envproteins with the signal peptide deleted for enhanced expression(prM-Env amino acids 216-794; also known as M-Env), adenovirus (Ad)vectors expressing this immunogen and purified PIV vaccines werecompared. All three types of vaccines are shown to induce ZIKV-specificneutralizing antibodies and protected both mice and rhesus monkeysagainst challenge with ZIKV strains from Brazil and Puerto Rico. Abbinket al., “Protective efficacy of multiple vaccine platforms against Zikavirus challenged in rhesus monkeys,” Science 353: 1129-32 (2016) and D.H. Barouch et al., “Prospects for a Zika Virus Vaccine,” Immunity 46:176-182 (2017); and Larocca et al., “Vaccine protection against Zikavirus from Brazil,” Nature 536: 474-478. In rhesus monkeys, the ZIKV PIVvaccine disclosed herein and the Ad vector-based vaccine both inducedneutralizing antibodies after a single immunization and proved moreimmunogenic than the DNA vaccine. The DNA vaccine did provide sufficientneutralizing antibodies only after two immunizations, which is a lesspreferred method of immunizing subjects when trying to reduce a viraloutbreak. D. H. Barouch et al. (2017).

Example 5. ZIKV PIV Performance in Human Trials

Five trials constitute the phase 1 clinical development program for ZPIVand were designed to explore different the safety and immunogenicityperformance across a variety of potential use scenarios.

Trial 1

-   -   ZIKV PIV (ZPIV) is provided to individuals with or without        previous exposure to yellow fever and Japanese encephalitis        vaccines to assess safety and immunogenicity of ZPIV in        scenarios where people will have pre-existing immunity to these        flaviviruses through vaccination; these data could also be        reasonably extrapolated to natural exposures as well

Trial 2

-   -   ZPIV is explored across a range of antigen doses in an attempt        to define the optimal dose of antigen as well as lower limits of        antigen concentration required to effectively immunize a        recipient

Trial 3

-   -   ZPIV is explored across a range of schedules in an attempt to        define the optimal dosing schedule to effectively immunize a        recipient and the potential requirement for booster doses to        create durable immune responses

Trial 4

-   -   ZPIV will be administered in an area in Puerto Rico with a high        rate of dengue virus priming as well as an areas having recently        experienced a large Zika outbreak to understand safety and        immunogencity performance in scenarios where dengue and Zika are        potentially endemic.

Trial 5

-   -   ZPIV will be administered in sequential, heterologous prime        boost scenarios with the NIH DNA vaccine candidate to understand        safety and potentially advantageous immunogenicity performance        characteristics

Data was reviewed for the ZPIV recipients without known previousexposure through natural infection or vaccination to Zika or otherflaviviruses. Two doses were administered at 0 and 28 days, 5 μg perdose, and adjuvanted with alum. Safety and immunogenicity data werecollected out to day 57. A total of 67 individuals were in this group,with 55 receiving ZPIV and 12 receiving placebo. Volunteers representenrollment from Trial Groups 2, 1 and 3 respectively.

A relatively equal number of male and female volunteers were enrolled;52 and 48%, respectively. The preponderance of volunteers were white(71%) or black (19%). The mean age was 31.5 years with a decline in themean age from Trial Group 2 (33.3) to Trial Group 1 (30.9) to TrialGroup 3 (27.9).

There were no deaths, serious adverse events related to vaccination, ordis-enrollments related to an adverse event. There were no severe localadverse events (pain, redness, swelling at the site of injection). Mostlocal adverse events were infrequent in occurrence and mild in severity.Pain and tenderness at the injection site occurred more frequently andwere overwhelmingly mild in severity. Any systemic sign or symptom wasreported in a majority of enrolled volunteers with the majority beingmild (49.2%) and a smaller proportion being graded as moderate (14.9%)or severe (1.5%). A single (1.5%) volunteer reported nausea and/orvomiting; there were no other severe systemic signs or symptoms. Insummary, despite not knowing which local or systemic signs or symptomswere reported by placebo or vaccine recipients, the safety profile inthese 67 volunteers is acceptable, comparable to many currently licensedvaccines, and supports advancing clinical development.

Immunogenicity data was generated through the use of microneutralizationassay and the titers reported indicating the readout representing thetiter at which 50% of the control virus is neutralized (MN50). Thecutoff for determining a vaccine take (seroconversion) is a MN titer of1:10. Titers are measured—28 days following the second dose of vaccine(study day 57). The MN50 titer found to be protective in mice andnon-human primate studies ranges from 1:10-1:100, respectively. Forcontext, the neutralizing antibody titers accepted by the regulatoryagencies as correlates or surrogates of protection for licensedflavivirus vaccines against yellow fever, Japanese encephalitis (JE),and tick borne encephalitis ranges from 1:5-1:10.

Seroconversion measured by the MN50 assay, >1:10 titer, at study day 57was 92% across the Trial 2 Group, Trial 3 Group, and Trial 1 Group studysites with a range of site specific seroconversion rates of Trial 2Group 92%, Trial 1 Group 88%, and Trial 3 Group 100%. Seroconversionwith a titer cutoff of 1:100 (protective titer in non-human primates)was Trial Group 2 65%, Trial Group 1 59%, and Trial Group 3 100%;overall rate of 69%. Antibody kinetics based on MN50 titers indicating aslight rise in antibody after the first dose of vaccine with mean titersremaining below the 1:10 titer cutoff and then a robust and brisk risein antibody titer for the Trial Group 2 and Trial Group 3 volunteersfollowing dose two. Peak titers for Trial Group 1 and Trial Group 3volunteers occur on or about study day 43 (2 weeks post dose two) andare between 500 and 1000. There is a gradual decline in titer betweenday 43 and day 57 with the final data point indicating titers between˜200-900. The Trial Group 1 volunteers have a distinct kinetic curvefollowing dose two with a gradual rise and no decline in titer peakingat ˜100 at day 57.

Detailed antibody data are provided in the table below representing sitespecific geometric mean titers and the confidence interval around thesetiters based on the day of collection and the study site where thecollection occurred. Peak titers are also represented.

5 mcg ZPIV Placebo Time 16-0033 16-0062 Z0001 All All Point^(a)Statistic (N = 25) (N = 20) (N = 10) (N = 55) (N = 12) Day 1 N* 25 20 1055 12   GMT 5.0 5.0 5.0 5.0 5.0 95% CI — — — — — Day 15 N* 25 — 10 357   GMT 5.0 — 8.6 5.8 5.0 95% CI — —  2.5, 28.8 4.3, 8.0 — Day 29 N* 2520 10 55 12   GMT 5.5 8.5 8.7 7.0 5.0 95% CI 4.5, 6.8 4.7, 15.3  2.5,30.4 5.2, 9.5 — Day 43 N* 25 — 10 35 7   GMT 316.9 — 983.3 437.9 5.0 95%CI 152.9, 656.6 —  425.4, 2272.5 245.7, 780.6 — Day 57 N* 25 17 9 5112   GMT 142.9 100.8 820.6 173.1 5.0 95% CI  70.3, 290.4 39.7, 255.7 357.1, 1885.8 104.6, 286.5 — Peak Titer N* 25 20 10 55 12   GMT 345.664.2 1061.7 229.8 5.0 95% CI 166.4, 718.0 25.3, 163.2  452.8, 2489.2132.6, 398.4 —

Based on the disclosure and the above data the compositions includinginactivated ZIKV demonstrate that the compositions are immunogenic andthat vaccines comprising inactivated ZIKV are protective againstinfection with ZIKV.

The available immunogenicity data for the ZPIV indicates the candidateis moderate to highly immunogenic following two doses measured out tostudy day 57. When aggregated across the sites, titers in the majorityof the vaccinated population exceed what is believed to a protectivetiter based on pre-clinical animal studies. To further support thiscontention, BIDMC completed a passive transfer study using purifiedantibody collected from the sera of human vaccine recipients. Nine (9)animals were fully protected by the human antibody following challenge,1 was partially protected, and 2 were not protected; these data trackwith the randomization scheme of 10:2 vaccine:placebo. The ZPIV dataalso tracks with known correlates and surrogates of protection forcurrently licensed flavivirus vaccines. Completion of the studies isrequired to further define immune response durability and understand ifcurrent variations is response across study sites is maintained whendata from all cohorts is collected and flavivirus primed and unprimedsubsets are defined.

ZPIV was well tolerated and safe in a small number of volunteers andmoderate to highly immunogenic. These data support proceeding toadvanced clinical development.

Incorporation by Reference

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific aspects of the subject disclosure have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the disclosure will become apparent to those skilled inthe art upon review of this specification and the claims below. The fullscope of the disclosure should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

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What is claimed is:
 1. A purified, inactivated, immunogenic ZIKV.
 2. Thepurified, inactivated, immunogenic ZIKV of claim 1, wherein the ZIKV isderived from PRVABC59.
 3. An immunogenic composition comprising apurified inactivated ZIKV of claim 1 and a pharmaceutically acceptableadjuvant.
 4. The immunogenic composition of claim 3, wherein theacceptable adjuvant is alum.
 5. The immunogenic composition of claim 3,wherein the purified inactivated ZIKV is derived from Puerto RicoPRVABC59, Thailand SV0127/14, Philippine COC C 0740, or BrazilFortaleza/2015, or other suitable strains.
 6. A vaccine comprising thepurified, inactivated, immunogenic ZIKV of claim 1 and apharmaceutically acceptable adjuvant.
 7. The vaccine of claim 6, whereinthe pharmaceutically acceptable adjuvant is alum.
 8. The vaccine ofclaim 7, wherein the purified inactivated ZIKV is derived from ZIKVPRVABC59.
 9. The vaccine of claim 6, wherein the purified inactivatedimmunogenic ZIKV is derived from Puerto Rico PRVABC59, ThailandSVO127/14, Philippine COC C 0740, or Brazil Fortaleza/2015, or othersuitable strains.
 10. A method of producing antibodies which recognizeZIKV in a host comprising administering to the host a compositioncomprising the immunogenic composition of claim
 3. 11. A method ofinducing a protective immune response against a Zika virus (ZIKV) in asubject, comprising the step of administering to the subject the vaccineof claim
 6. 12. The method of claim 11, wherein the administering is viaintramuscular injection, intradermal injection, subcutaneous injection,intravenous injection, oral administering, or intranasal administering.13. A method treating or alleviating symptoms of ZIKV in a subject,comprising the step of administering to the subject the immunogeniccomposition of claim
 3. 14. A medicament comprising the immunogeniccomposition of claim
 3. 15. A medicament comprising the vaccine of claim6.
 16. A method of generating a purified inactivated ZIKV comprising thesteps of: i) inoculating a cell culture with an amount of a ZIKV strain;ii) growing the inoculated virus in cell culture; iii) harvesting andisolating virus fluids from the inoculated cell culture to prepare aZika virus concentrate; iv) purifying the ZIKV concentrate; v)inactivating the purified ZIKV; and vi) recovering the purified,inactivated ZIKV.
 17. The method of claim 16, wherein the purified ZIKVis inactivated by contacting the ZIKV with a chemical inactivatingagent.
 18. The method of claim 17, wherein the chemical inactivatingagent is formalin, beta-propiolactone, or hydrogen peroxide.
 19. Themethod of claim 17, wherein the ZIKV strain is derived from Puerto RicoPRVABC59, Thailand SV0127/14, Philippine COC C 0740, or BrazilFortaleza/2015, or other suitable strains.
 20. A purified inactivatedZIKV produced by the method of claim
 16. 21. (canceled)